SPARC-derived tumor rejection antigenic peptides and medicaments comprising the same

ABSTRACT

It is an objective of the present invention to identify SPARC protein-derived peptides that are able to induce human killer T cells and helper T cells having cytotoxic activity to tumors, and to provide a means for carrying out a tumor immunotherapy of patients with various types of cancers overexpressing SPARC. The present invention provides a peptide of any of the following:
     (A) a peptide which consists of the amino acid sequence as shown in any one of SEQ ID NOS: 1 to 3; or   (B) a peptide which consists of an amino acid sequence comprising a substitution or addition of one or several amino acids with respect to the peptide consisting of the amino acid sequence as shown in any one of SEQ ID NOS: 1 to 3, and which has capacity to induce cytotoxic (killer) T cells.

TECHNICAL FIELD

The present invention relates to novel peptides that are effective as avaccine for cancers highly expressing SPARC, such as stomach cancer,pancreatic cancer or malignant melanoma (melanoma), and medicamentscomprising the aforementioned peptides used for treating and/orpreventing tumors.

BACKGROUND ART

When compared with Western countries, the morbidity of stomach cancer ishigh in Asian countries such as Japan and China. As a result ofdiffusion of medical check-ups, the widespread use of digestiveendoscopes, and the development of inspection techniques, it has becomepossible to detect stomach cancer at an early stage, and thus the numberof patients suffering from this cancer has been decreasing.Nevertheless, stomach cancer is still the second cause of death due tomalignant neoplasm in the Japanese population. Thus, stomach cancer isstill a main cause of death. Among several types of stomach cancers,diffuse (scirrhous) stomach cancer occurs in young people when comparedwith another type of stomach cancer (adenocarcinoma). Such diffuse(scirrhous) stomach cancer tends to make rapid progress, and distantmetastasis or peritoneal metastasis frequently occurs, thereby resultingin poor prognosis. In many cases of scirrhous stomach cancer, it hasalready become impossible to carry out surgical excision at the time ofdiagnosis. Even if it is still possible to excise the tumor, the canceroften recurs after the treatment. Accordingly, it is highly desired toestablish a novel treatment method.

The death toll from pancreatic cancer tends to increase in Japan. 21,148people died due to pancreatic cancer in 2003. At present, suchpancreatic cancer makes up 6.8% of the cancers that cause death inJapan. That is, pancreatic cancer ranks fifth cause of death after lungcancer, stomach cancer, colon cancer, and liver cancer. The worlddemographic data was analyzed, and the age-adjusted mortality rate,which is used for comparison among the mortality rates of populationswith different age structures, was calculated. As a result, in 2000, inthe case of 100,000 males, 8.6 people died due to pancreatic cancer inJapan, whereas 7.3 people died in the United States and 6.3 to 7.0people died in the United Kingdom due to the same type of cancer. In thecase of 100,000 females, 4.9 people died due to pancreatic cancer inJapan, whereas 5.3 people died in the United States and 4.8 to 5.1people died in the United Kingdom due to the same type of cancer. Thus,the mortality rate due to pancreatic cancer in Japan has become the samelevel as those of Western countries.

Taking into consideration the age-adjusted rate in 2000 (100,000 peopleof the world's population), in the case of males, 8.6 people died due topancreatic cancer in Japan, whereas 7.3 people died in the United Statesand 6.3 to 7.0 people died in the United Kingdom due to the same type ofcancer. In addition, in the case of females, 4.9 people died due topancreatic cancer in Japan, whereas 5.3 people died in the United Statesand 4.8 to 5.1 people died in the United Kingdom due to the same type ofcancer. Thus, the mortality rate due to pancreatic cancer in Japan hasbecome the same level as those of Western countries. In spite of thedevelopment of diagnostic imaging, at present, approximately 40% of thetotal Japanese patients with pancreatic cancer suffer from progressivepancreatic cancer involving distant metastasis, and further, there arealso many cases where the cancer is discovered after it has reached alocally-advanced cancer stage, at which tumor cannot be excised. The5-year relative survival rate of total patients with pancreatic canceris 4.3% in the diagnosis cases in 1996. Although this rate tends to behigher than the conventional survival rate (2% to 3%), it is still low.Regarding factors of developing pancreatic cancer, it has been suggestedthat various factors including life habit such as smoking, adiposis,meals, alcohol drinking, and coffee use, as well as chronicpancreatitis, diabetes, genetic factor, etc. are involved in the onsetof pancreatic cancer.

Pancreatic cancer does not have specific symptoms, and thus, in manycases, the cancer has already progressed when certain symptoms appear.As a result, the 5-year survival rate of total patients is 5% or less,and prognosis after the diagnosis is extremely low. Due to difficulty inthe diagnosis of pancreatic cancer, the rate of this cancer as acausative disease of cancer death is gradually increased particularly inadvanced countries. Currently, multidisciplinary therapy including asurgical excision as a main treatment, a radiotherapy, and achemotherapy, has been carried out. However, no drastic improvement oftherapeutic effects can be obtained, and thus, the establishment of anovel therapeutic strategy is urgently necessary.

Melanoma is one type of skin cancer, which is often called malignantmelanoma. Among several types of skin cancers, melanoma is highly likelyto become infiltrative and metastatic and has the highest grade ofmalignancy, and thus it is greatly feared. Among cells that constituteskin, several cells generate melanin pigment. Such cells are calledmelanocytes. When such melanocytes become cancerous, melanoma occurs. Inaddition, the frequency of occurrence of melanoma has been increasing,particularly among Caucasians, as a result of an increase in exposure toultraviolet rays due to a reduction in the ozone layer in the atmospherecaused by environmental destruction.

In Japan, the incidence of melanoma varies from 1.5 to 2 people in100,000 in the general population. Thus, it is estimated thatapproximately 1,500 to 2,000 people develop melanoma per year. On theother hand, in the Western countries, more than a dozen of peopledevelop melanoma in 100,000 in the general population. In particular, inAustralia, twenty or more people develop such melanoma in 100,000 in thegeneral population, and thus it is known that the incidence of melanomain Australia is the highest in the world. Under such circumstances,people who live in Europe, the United States, and Australia areinterested in melanoma, and they pay attention to the occurrence ofmelanoma. Furthermore, surprisingly, the occurrence of melanoma tends tobe increasing year after year in Japan as well as in foreign countries.According to recent studies, the annual death toll from melanoma isapproximately 450 in Japan. Melanoma develops regardless of age.However, the incidence of this disease increases for those over 40, andit is the highest for those in their 60's and 70's. The onset of thisdisease in childhood is extremely rare, but this does not mean that thedisease never develops in childhood. Recently, the occurrence ofmelanoma tends to be increasing in young patients in their 20's and30's. Melanoma develops regardless of sex, and both male and femalepatients suffer from this disease. In the case of Japanese patients, thesite at which melanoma is most likely to develop is the sole (the soleof the foot), and it accounts for 30% of all instances of melanoma. Ascharacteristics of Japanese patients, melanoma also develops in the footand the nail portions of the fingers. In addition, as in the case ofWestern patients, melanoma develops in all parts of the skin, such asthe body, hand, foot, face, and head.

At present, methods that can be applied to treat melanoma include asurgical therapy, a chemotherapy, and a radiotherapy. However, as atherapy for alleviating the symptoms of metastatic cancer or intractablecancer, to which the aforementioned therapy cannot be applied, animmunotherapy for enhancing the immunity of a cancer patient to cancerso as to suppress the growth of the cancer has become a focus ofattention. Such an immunotherapy is actually effective for somepatients.

On the other hand, with the development of molecular biology and tumorimmunology in recent years, it has been revealed that cytotoxic (killer)T cells and helper T cells recognize peptides generated by degradationof proteins highly and specifically expressed in cancer cells, which arepresented on the surfaces of the cancer cells or antigen-presentingcells via HLA molecules, and that they exhibit an immune reaction fordestroying such cancer cells. Moreover, a large number of tumorantigenic proteins and peptides derived from them, which stimulate suchan immune reaction for attacking cancers, have been identified, andclinical application of an antigen-specific tumor immunotherapy has beenadvanced.

HLA class I molecules are expressed on the surfaces of all nucleatedcells in a body. Proteins generated in cytoplasms and nuclei providepeptides generated as a result of being degraded in cells, and they areexpressed on the surfaces of such cells. On the surfaces of normalcells, peptides derived from normal autologous proteins bind to HLAclass I molecules, and T cells of the immune system neither recognizenor destroy such peptides bound to HLA class I molecules. On the otherhand, in a process in which cancer cells are converted to a cancer, suchcancer cells may express large amounts of proteins, which are hardlyexpressed or are only expressed in small amounts on normal cells. If apeptide generated by degradation in the cytoplasm of such a protein thatis highly expressed specifically in a cancer cell binds to an HLA classI molecule and is expressed on the surface of such a cancer cell, akiller T cell recognizes the peptide and destroys only the cancer cell.In addition, by immunizing such a cancer-specific antigen or peptide toan individual body, it is possible to destroy cancer cells and suppressthe growth of a cancer, without impairing normal cells. This is referredto as cancer immunotherapy using a cancer-specific antigen. Moreover, anHLA class II molecule is mainly expressed on the surface of anantigen-presenting cell. Such an HLA class II molecule binds peptidesderived from a cancer-specific antigen generated by incorporating thecancer-specific antigen from outside the cell and degrating it in thecell, and it is expressed on the surface of the cell. A helper T cell,which has recognized the peptides bound to HLA class II molecule, isactivated to generate various cytokines that activate otherimmunocompetent cells, so as to induce or reinforce an immune reactionagainst a tumor.

Thus, if an immunotherapy targeting an antigen specifically expressingat a high level in such a cancer can be developed, it can provide atherapeutic method effectively eliminating the cancer alone, withoutimpairing normal autologous organs. Moreover, it is anticipated thatsuch an immunotherapy can provide a therapeutic method applicable topatients suffering from a terminal-stage cancer, for whom no othertreatments can be implemented. Furthermore, if a cancer-specific antigenand peptide are administered in the form of a vaccine to a human havinga high risk of developing such a cancer, there is a possibility that theonset of the cancer can be prevented.

First, the present inventors have performed a genome-wide geneexpression analysis. They have analyzed 23,040 types of genes in stomachcancer tissues and normal tissues utilizing a cDNA microarray. As aresult, in 11 out of 20 cases of patients with diffuse infiltrativestomach cancer, the inventors have identified Secreted protein acidicand rich in cysteine (SPARC), which is a gene highly expressed instomach cancer tissues, and the expression level of which is 5 or moretimes higher than that of normal tissues (130,000 times higher onaverage) (FIG. 1). The SPARC gene is expressed at a low level also innormal adipose tissues, mammary gland, ovary, spinal cord, testis,uterus, placenta, etc. However, the expression level of the SPARC genein any of the aforementioned organs is lower than that of normal gastricmucous membrane by a factor of 5 times or less (FIG. 2).

Other researchers had already reported that SPARC is not only highlyexpressed in diffuse infiltrative stomach cancer but it is alsoexpressed in pancreatic cancer and melanoma. Moreover, the presentinventors have found that SPARC is secreted in the serum of melanomapatients, and that SPARC can be a useful tumor marker particularly forthe early detection of melanoma (Japanese Patent Application No.2004-303688; and Clinical Cancer Research 11: 8079-8088, 2005).

SPARC is a 43-KD acidic secretory protein consisting of 286 amino acids.This protein is rich in cysteine, and it moves to the nucleus during thecell division phase. In addition, SPARC controls the interaction betweenan extracellular matrix protein and a cell, so that it can be alsoassociated with the control of cell growth. Since SPARC is expressed inosteoblasts, thrombocytes, and wound areas, it is considered that thisprotein is associated with the repair and reconstruction of tissues.Furthermore, it has also reported that SPARC is highly expressed also incancers such as melanoma or osteosarcoma and in the interstitial cellsof tumors, and that the expression of SPARC correlates with theprognosis, infiltration or metastasis of tumors.

-   [Non-Patent Document 1] Ikuta Y et al., Clinical Cancer Research 11:    8079-8088, 2005.-   [Patent Document 1] Japanese Patent Application No. 2004-303688

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

It is an objective of the present invention to develop a method forenhancing the immunity of a patient suffering from metastatic cancer orintractable cancer, to which surgical therapy, chemotherapy andradiotherapy are used as treatments for tumors highly expressing SPARC,such as diffuse infiltrative stomach cancer, pancreatic cancer ormelanoma, can hardly be applied, so as to alleviate the symptoms of suchcancers and so as to carry out an immunotherapy suppressing the growthof such cancers. That is to say, it is an objective of the presentinvention to identify a peptide derived from an SPARC protein that isoverexpressed in cancer tissue, which is able to induce a strong immuneresponse to the aforementioned cancers without causing harmfulphenomenon to cancer patients, and to apply the identified peptide to atumor immunotherapy. That is, it is an objective of the presentinvention to identify a SPARC protein-derived peptide that is able toinduce human killer T cells and helper T cells having reactivity totumors, and to provide a means for carrying out a tumor immunotherapy onpatients with various types of cancers highly expressing SPARC.

Means for Solving the Problems

The present inventors have identified a gene, SPARC, that is highlyexpressed in diffuse infiltrative stomach cancer, by performing cDNAmicroarray analysis on the aforementioned stomach cancer and variousnormal tissues. The expression of SPARC is observed also in severaltypes of normal tissues. However, the expression level of SPARC innormal tissues is significantly lower than that in cancerous tissues. Inorder to examine the presence or absence of the induction of antitumorimmunity by SPARC-specific killer T cells, there were utilized BALB/cmice that express mouse K^(d) molecules, the characteristics of theamino acid sequence of the bound peptide of which are identical to thoseof HLA-A24 that is the most frequent HLA class I allele in the Japanesepopulation. Human SPARC has 95% homologous amino acid sequence incomparison to mouse SPARC. Thereby peptides consisting of amino acidsequences shared between the humans and the mice, and having a bindingmotif shared by human HLA-A24 and mouse K^(d), were synthesized.Thereafter, BALB/c mice (K^(d)-expressed) were immunized with bonemarrow-derived dendritic cells, on which such a peptide mixture had beenloaded, and the inventors investigated whether killer T cells reactiveto SPARC-expressing cancer cells are induced or not. Moreover, withregard to mice that had been pre-treated by the same immunization methodas described above, the inventors investigated whether the growth of thetransplanted cancer cells expressing mouse SPARC is suppressed and thesurvival time of the mice is prolonged or not. Furthermore, whether ornot a harmful phenomenon appears together with the occurrence of anautoimmune phenomenon in the peptide-immunized mice was also examined.As a result, it was found that the peptide having the amino acidsequence as shown in any one of SEQ ID NOS: 1 to 3 is able to inducekiller T cells that destroy cancer cells expressing SPARC. Furthermore,the growth of the transplanted mouse cancer cells expressing SPARC issuppressed in mice immunized with the aforementioned peptide and thusthe survival time of the mice is prolonged. The present invention hasbeen completed based on these findings.

The present invention provides the following invention.

(1) A peptide of any of the following:

(A) a peptide which consists of the amino acid sequence as shown in anyone of SEQ ID NOS: 1 to 3; or

(B) a peptide which consists of an amino acid sequence comprising asubstitution or addition of one or several amino acids with respect tothe peptide consisting of the amino acid sequence as shown in any one ofSEQ ID NOS: 1 to 3, and which has ability to induce cytotoxic (killer) Tcells.(2) An immune inducing agent for cancers, which comprises at least onetype of the peptide of (1).(3) A medicament for treating and/or preventing tumors, which comprisesat least one type of the peptide of (1).(4) An agent for inducing antigen-presenting cells having high abilityto induce tumor-reactive T cells, which comprises at least one type ofthe peptide of (1).(5) An agent for inducing tumor-reactive T cells, which comprises atleast one type of the peptide of (1).(6) An agent for inducing antigen-presenting cells having high abilityto induce tumor-reactive T cells, which comprises a gene encoding for apeptide of any of the following:(A) a peptide which consists of the amino acid sequence as shown in anyone of SEQ ID NOS: 1 to 3; or(B) a peptide which consists of an amino acid sequence comprising asubstitution or addition of one or several amino acids with respect tothe peptide consisting of the amino acid sequence as shown in any one ofSEQ ID NOS: 1 to 3, and which has ability to induce killer T cells.(7) An antibody against the peptide of (1).(8) A killer T cell, a helper T cell, or an immunocyte populationcomprising such cells, which is induced using the peptide of (1).(9) An antigen-presenting cell, which presents a complex of an HLAmolecule and the peptide of (1).(10) The antigen-presenting cell of (9), which is induced using theagent of (4) or (6).

BEST MODE FOR CARRYING OUT THE INVENTION (1) Peptide of the PresentInvention, and Immune Inducing Agent for Cancers Comprising the Same

The peptide of the present invention is described in the following:

(A) a peptide which consists of the amino acid sequence as shown in anyone of SEQ ID NOS: 1 to 3; or

(B) a peptide which consists of an amino acid sequence comprising asubstitution or addition of one or several amino acids with respect tothe amino acid sequence as shown in any one of SEQ ID NOS: 1 to 3, andwhich has ability to induce killer T cells.

The term “peptide having ability to induce cytotoxic T cells” is used inthe present specification to mean a peptide having a activity tostimulate tumor-reactive killer T cells.

A method for obtaining/producing the peptide of the present invention isnot particularly limited. Either a chemically synthesized peptide, or arecombinant peptide produced by genetic recombination, may be used.

When a chemically synthesized peptide is obtained, the peptide of thepresent invention can be synthesized by a chemical synthesis method suchas an Fmoc method (fluorenylmethyloxycarbonyl method) or a tBoc method(t-butyloxycarbonyl method), for example. In addition, the peptide ofthe present invention can also be synthesized using various types ofcommercially available peptide synthesizers.

When the peptide of the present invention is produced in the form of arecombinant protein, DNA having a nucleotide sequence encoding theaforementioned peptide, a mutant thereof, or a homologue thereof isobtained, and it is then introduced into a preferred expression system,so as to produce the peptide of the present invention.

As an expression vector, a vector capable of autonomously replicating ina host cell or capable of being incorporated into the chromosome of ahost cell may preferably be used. An expression vector comprising apromoter at a position capable of expressing a gene encoding for thepeptide is used. In addition, a transformant having a gene encoding forthe peptide of the present invention can be produced by introducing theaforementioned expression vector into a host. As a host, any one of abacterium, yeast, an animal cell, and an insect cell may be used. Anexpression vector may be introduced into a host according to a knownmethod, depending on the type of such a host.

In the present invention, the transformant as produced above iscultured, and the peptide of the present invention is then generated andaccumulated in a culture. Thereafter, the peptide of the presentinvention is collected from the culture, so as to isolate a recombinantpeptide.

When such a transformant is a prokaryote such as Escherichia coli or aeukaryote such as yeast, a medium used for culturing such microorganismsmay be either a natural medium or a synthetic medium, as long as itcontains a carbon source, a nitrogen source, inorganic salts, and thelike that can be assimilated by the aforementioned microorganisms, andit is able to efficiently carry out the culture of the transformant.Moreover, such culture may be carried out under conditions that arecommonly applied for culturing the aforementioned microorganisms. Aftercompletion of the culture, the peptide of the present invention may beisolated and purified from the culture of the transformant according toa common method of isolating and purifying a peptide.

The term “one or several amino acids” is used in the presentspecification to mean generally 1 to 10 amino acids, preferably 1 to 8amino acids, more preferably 1 to 5 amino acids, and particularlypreferably 1 to 3 amino acids (for example, 1, 2 or 3 amino acids).

A peptide consisting of an amino acid sequence comprising a substitutionor addition of one or several amino acids with respect to the peptideconsisting of the amino acid sequence as shown in any one of SEQ ID NOS:1 to 3 can be appropriately produced or acquired by persons skilled inthe art based on information regarding the amino acid sequence as shownin any one of SEQ ID NOS: 1 to 3. That is to say, a peptide whichconsists of an amino acid sequence comprising a substitution or additionof one or several amino acids with respect to the amino acid sequence asshown in any one of SEQ ID NOS: 1 to 3 and which has ability to inducecytotoxic T cells, can be produced by any given method known to personsskilled in the art, such as the aforementioned chemical synthesis,genetic engineering means, or mutagenesis. For example, site-directedmutagenesis which is a genetic engineering means is useful because it isa means for introducing a specific mutation into a specific position.Such site-directed mutagenesis can be carried out by a method describedin Molecular Cloning: A laboratory Manual, 2^(nd) Ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989 (hereinafterabbreviated as Molecular Cloning 2^(nd) Ed.), Current Protocols inMolecular Biology, Supplement 1 to 38, John Wiley & Sons (1987-1997)(hereinafter abbreviated as Current Protocols in Molecular Biology),etc.

As described later in examples, the aforementioned peptide of thepresent invention is able to induce immunity against cancers. Thus, thepresent invention provides an immune inducing agent for cancers, whichcomprises the peptide of the present invention.

The immune inducing agent of the present invention used for cancers isused in vitro, ex vivo, or in vivo, and preferably ex vivo, so that itcan induce killer T cells, helper T cells, or an immunocyte populationcomprising such cells, thereby imparting immunity against cancers.

(2) Antibody of the Present Invention

The present invention also relates to an antibody that recognizes a partor whole of the aforementioned peptide of the present invention as anepitope (antigen), and a killer T cell induced by ex vivo or in vitrostimulation using the aforementioned peptide. In general, it has beenknown that such a killer T cell exhibits an antitumor activity that isstronger than that of an antibody.

The antibody of the present invention may be either a polyclonalantibody or a monoclonal antibody. Such an antibody can be produced by acommon method.

For example, a polyclonal antibody can be produced by immunizing amammal or Ayes with the peptide of the present invention used asantigen, then collecting blood from the mammal or Ayes, and thenseparating and purifying an antibody from the collected blood. Forexample, mammals or Ayes, such as a mouse, a hamster, a guinea pig, achicken, a rat, a rabbit, a canine, a goat, a sheep, a bovine or ahorse, can be immunized. Such an immunization method is known to personsskilled in the art. For example, an antigen may be administered 2 or 3times at intervals of 7 to 30 days. As a dosage, approximately 0.05 to 2mg of antigen can be administered once, for example. An administrationroute is not particularly limited, and subcutaneous administration,intracutaneous administration, intraperitoneal administration,intravenous administration, intramuscular administration, etc. can beselected, as appropriate. Moreover, an antigen can be dissolved in asuitable buffer containing an adjuvant, for example, in a suitablebuffer containing a commonly used adjuvant such as a complete Freund'sadjuvant or aluminum hydroxide, and it can be used.

Thus the immunized mammal or Ayes is bred for a certain period of time.Thereafter, if the antibody titer increases, a booster can be carriedout using 100 to 1,000 μg of antigen, for example. One or two monthsafter the final immunization, blood is collected from the immunizedmammal or Ayes. The thus collected blood (polyclonal antiserum) is thenseparated and purified by an ordinary method including centrifugation,precipitation using ammonium sulfate or polyethylene glycol,chromatography such as gel filtration chromatography, ion exchangechromatography, or affinity chromatography, etc., so as to obtain apolyclonal antibody that recognizes the peptide of the presentinvention.

On the other hand, a monoclonal antibody can be obtained by preparing ahybridoma. For example, such a hybridoma can be obtained by cell fusionof an antibody-generating cell and a myeloma cell. A hybridoma thatgenerates the monoclonal antibody of the present invention can beobtained by the following cell fusion method.

As an antibody-generating cell, a spleen cell, a lymph node cell, a Blymphocyte, or the like obtained from the immunized animal is used. Asan antigen, the peptide of the present invention is used. As an animalto be immunized, a mouse, a rat, or the like can be used. An antigen isadministered to such an animal according to an ordinary method. Forexample, a suspension or emulsified liquid comprising an adjuvant suchas a complete Freund's adjuvant or incomplete Freund's adjuvant and thepeptide of the present invention used as an antigen is administered toan animal via intravenous administration, subcutaneous administration,intracutaneous administration, intraperitoneal administration, etc.,several times, so as to immunize the animal. Thereafter, anantibody-generating cell such as a spleen cell is obtained from theimmunized animal, and the thus obtained spleen cell is then fused with amyeloma cell according to a known method (G. Kohler et al., Nature, 256495 (1975)), thereby producing a hybridoma.

Examples of a myeloma cell strain used in cell fusion include a P3X63Ag8strain, a P3U1 strain and an Sp2/0 strain, in the case of a mouse. Whensuch cell fusion is carried out, a fusion promoter such as polyethyleneglycol or Sendai virus is used. For selection of a hybridoma aftercompletion of the cell fusion, a hypoxanthine aminopterin thymidine(HAT) medium is used according to an ordinary method. The hybridomaobtained as a result of the cell fusion is cloned by a limiting dilutionmethod. Further, as necessary, screening is carried out by an enzymeimmunoassay using the peptide of the present invention, so as to obtaina cell strain that generates a monoclonal antibody specificallyrecognizing the peptide of the present invention.

In order to produce a monoclonal antibody of interest from the thusobtained hybridoma, the hybridoma may be cultured by a common cellculture method or ascites formation method, and the monoclonal antibodyof interest may be then purified from the culture supernatant orascites. The monoclonal antibody may be purified from the culturesupernatant or ascites according to an ordinary method. For example,ammonium sulfate fractionation, gel filtration, ion exchangechromatography, affinity chromatography, and other methods may becombined as appropriate and used.

Moreover, the fragments of the aforementioned antibody are also includedin the scope of the present invention. Examples of such an antibodyfragment include an F(ab′)2 fragment and an Fab′ fragment.

(3) Killer T Cell, Helper T Cell, or Immunocyte Population Comprisingsuch Cells

The present invention also relates to a killer T cell, a helper T cellor an immunocyte population comprising such cells, which is induced byin vitro stimulation using the peptide of the present invention. Forexample, when peripheral blood lymphocytes or tumor-infiltratinglymphocytes are stimulated in vitro with the peptide of the presentinvention, activated T cells exhibiting tumor-reactivity are induced.Thus the activated T cells can be effectively used for an adoptiveimmunotherapy of cancer. Furthermore, the peptide of the presentinvention is allowed to be expressed in dendritic cells that are strongantigen-presenting cells in vivo or in vitro, and dendritic cellsexpressing the antigenic peptide are then administered so as to inducean immune response to tumors.

Preferably, a killer T cell, a helper T cell, or an immunocytepopulation comprising such cells can be induced by ex vivo or in vitrostimulation using the peptide of the present invention and animmunostimulator. Examples of such an immunostimulator used hereininclude a cell growth factor and a cytokine.

The thus obtained killer T cell, helper T cell, or immunocyte populationcomprising such cells is transferred into a body, so that tumor can besuppressed and that cancer can be prevented and/or treated.

Further, using the peptide of the present invention, a killer T cell, ahelper T cell, or an immunocyte population comprising such cells, whichis capable of suppressing tumor growth as described above, can beproduced. Accordingly, the present invention provides a cell culturesolution comprising tumor-reactive T cells and the peptide of thepresent invention. Using such a cell culture solution, a killer T cell,a helper T cell, or an immunocyte population comprising such cells,which is capable of suppressing tumor growth, can be produced. Stillfurther, the present invention also provides a cell culture kit forproducing a killer T cell, a helper T cell, or an immunocyte populationcomprising such cells, which comprises the aforementioned cell culturesolution and a cell culture vessel.

(4) Medicament of the Present Invention for Treating and/or PreventingTumor (Cancer Vaccine)

Since the peptide of the present invention is able to induce cancercell-specific killer T cells, it can be expected as an agent fortreating and/or preventing cancer. For example, bacteria such as BCG(Bacillus Calmette-GuErin) which was transformed with recombinant DNAproduced by incorporating a gene encoding for the peptide of the presentinvention into a suitable vector, or viruses such as vaccinia virus,into the genome of which DNA encoding for the peptide of the presentinvention has been incorporated, can be effectively used as a livevaccine for treating and/or preventing human cancers. It is to be notedthat the dosage and administration method of a cancer vaccine are thesame as those in the case of an ordinary smallpox vaccination or BCGvaccination.

That is to say, DNA encoding for the peptide of the present invention(which is used as is, or is in the form of plasmid DNA incorporated intoan expression vector), or a recombinant virus or recombinant bacteriacomprising the aforementioned DNA, can be administered as a cancervaccine to mammals including a human, directly or in a state where it isdispersed in an adjuvant. Likewise, the peptide of the present inventioncan also be administered as a cancer vaccine in a state where it isdispersed in an adjuvant.

Examples of an adjuvant used in the present invention include anincomplete Freund's adjuvant, BCC, trehalose dimycolate (TDM),lipopolysaccharide (LPS), an alum adjuvant, and a silica adjuvant. Fromthe viewpoint of ability to induce antibody, an incomplete Freund'sadjuvant (IFA) is preferably used.

The type of a cancer is not particularly in the present specification.Specific examples of a cancer include stomach cancer, colon cancer,esophageal cancer, pancreatic cancer, hepatic cancer, gallbladdercancer, cholangiocarcinoma, lung cancer, breast cancer, thyroid cancer,melanoma (malignant melanoma), skin cancer, osteosarcoma,pheochromocytoma, head and neck cancer, brain tumor, chronic myelogenousleukemia, acute myelogenous leukemia, malignant lymphoma, kidney cancer,bladder cancer, prostatic cancer, testicular cancer, uterine cancer,ovarian cancer, and soft tissue sarcoma. Of these, cancers that highlyexpress SPARC, such as stomach cancer (in particular, diffuseinfiltrative stomach cancer), pancreatic cancer, and melanoma (malignantmelanoma), are typical examples.

The peptide of the present invention acts as a T cell epitope to inducea cancer cell-specific killer T cell or helper T cell. Thus, the peptideof the present invention is useful as an agent for preventing and/ortreating human cancers. In addition, if the antibody of the presentinvention is able to inhibit the activity of SPARC as a cancer antigen,it is also useful as an agent for preventing and/or treating humancancers. As an actual usage, the peptide or antibody of the presentinvention can be administered as an injection product, directly ortogether with a pharmaceutically acceptable carrier and/or diluent, andas necessary, also together with the below-mentioned auxiliarysubstances. Moreover, the peptide or antibody of the present inventioncan also be administered by a method such as spraying, via transdermalabsorption through mucosa. The term “carrier” is used herein to meanhuman serum albumin, for example. In addition, as a diluent, PBS,distilled water, or the like can be used.

As a dosage, the peptide or antibody of the present invention can beadministered within the range between 0.01 and 100 mg per adult peradministration. However, the dosage is not limited to the aforementionedrange. The dosage form is not particularly limited, either. Afreeze-dried product, or a granule produced by adding an excipient suchas sugar, may also be available.

Examples of an auxiliary substance, which may be added to the agent ofthe present invention to enhance tumor-reactive T cell-inducingactivity, include: muramyl-dipeptide (MDP); bacterial components such asBCG bacteria; ISCOM described in Nature, vol. 344, p. 873 (1990);saponin QS-21 described in J. Immunol. vol. 148, p. 1438 (1992);liposome; and aluminum oxide. Further, immunostimulators such aslenthinan, schizophyllan, or Picibanil may also be used as auxiliarysubstances. Other examples of products used herein as auxiliarysubstances include: cytokines for enhancing the growth ordifferentiation of T cells, such as IL-2, IL-4, IL-12, IL-1, IL-6, orTNF; α galactosylceramide for activating NKT cells; CpG that binds to aToll-like receptor to activate a natural immune system; andlipopolysaccharide (LPS).

Furthermore, the aforementioned antigen peptide is added in vitro tocells collected from a HLA-A24-positive patient or allogeneic cellsisolated from anyone else who is HLA-A24-positive, followed by antigenpresentation of the cells. Thereafter, the cells are administered intothe blood vessel of the patient, so that killer T cells can beeffectively induced in the body of the patient. Further, the presentpeptide is added to the peripheral blood lymphocytes of a patient, andthe obtained mixture is then cultured in vitro. Thereby, killer T cellscan be induced in vitro, and they can be then returned to the bloodvessel of the patient. Such a therapy involving cell transfer hasalready been carried out as a method for treating cancers, and thus itis a method well known to persons skilled in the art.

By introducing the peptide of the present invention into a body,tumor-reactive T cells are induced, and as a result, an antitumor effectcan be anticipated. Moreover, when lymphocytes are stimulated by thepeptide of the present invention ex vivo or in vitro, activated T cellsare induced. The activated T cells are injected into an affected area.Thus, this technique can be effectively used for an adoptiveimmunotherapy.

The present invention will be further described in the followingexamples. However, these examples are not intended to limit the scope ofthe present invention.

EXAMPLES Example 1 (1) cDNA Microarray Analysis

With regard to the excised cancerous tissue of a patient with diffuseinfiltrative stomach cancer, a cancerous tissue was distinguished fromnon-cancerous tissues by Laser capture microdissection, and both thecancerous and non-cancerous portions were then cut out. Thereafter, RNAwas extracted from each tissue. When cDNA was synthesized from such RNAby a reverse transcription reaction, cDNA generated from canceroustissue was fluorescently labeled with Cy5, and cDNA generated fromnon-cancerous tissue was fluorescently labeled with Cy3. Subsequently,the two cDNA preparations were mixed to obtain target DNA. Hybridizationwas carried out on a slide glass on which probe cDNA had been arrayed,and non-specific bonds were then eliminated by washing. Thereafter,fluorescence images obtained after the hybridization were captured usinga CCD camera or a fluorescence scanner, and they were then displayedwith false colors (Cy5: red; Cy3: green). At the same time, the ratio ofthe two types of fluorescence intensity (R/G) was calculated, and it wasindicated as a gene expression profile. Moreover, the expression of23,040 types of genes in the cancerous and non-cancerous portions of 20patients with diffuse infiltrative stomach cancer was subjected tocomparative investigation, and 15 types of genes of which expressionratio in cancerous tissue/non-cancerous tissue was 5 or greater wereselected (FIG. 1).

Subsequently, the expression profile of the 23,040 types of genes in thenormal tissues of 29 organs (including 4 embryonal organs) was analyzed,and genes of which expression level was low in such normal organs wereselected. In the case of SPARC, which we selected as a tumor antigen inthis experiment, the expression ratio of cancerous/non-cancerousportions was 5 or greater (133,359 times on average) in 11 out of 20patients with diffuse infiltrative stomach cancer. SPARC was expressedin some normal tissues, but the expression levels in such anon-cancerous tissues were significantly lower than that in a canceroustissues (FIG. 2).

Example 2 (2) Selection of Human HLA-A24- and Mouse K^(d)-RestrictedSPARC Peptide Repertoires

In human and mouse SPARC having a homology of 95%, the followingpeptides were selected from the fragment of SPARC of which amino acidsequences were shared between humans and mice. The total amino acidsequences of SPARC molecules were searched based on known informationregarding amino acid sequences commonly observed in peptides havingstrong binding affinity to both human HLA-A24 and mouse K^(d) molecules.The repertoires of peptides that were assumed to have high bindingaffinity for the two types of molecules were selected by using acomputer program. As a result, 4 types of peptides were selected ascandidate peptides (Table 1).

TABLE 1 SPARC-derived peptides possibly having binding affinity to bothhuman HLA-A24 and mouse K^(d) molecules Start Binding score PeptideSequence position A24 K^(d) SPARC- K^(d)-1 DYIGPCKYI 143 75 400 SPARC-K^(d)-2 HFFATKCTL 123 20 1382  SPARC- K^(d)-3 EFPLRMRDWL 161 30 960SPARC- K^(d)-4 MYIFPVHWQF 225 210  120 Predicted by http://bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform SPARC Kd-1: SEQID NO: 1 SPARC Kd-3: SEQ ID NO: 2 SPARC Kd-4: SEQ ID NO: 3

(3) Dendritic Cell (DC) Vaccine

Bone marrow-derived dendritic cells (BM-DC) were induced from bonemarrow cells derived from BALBc bone marrow by using GM-CSF as describedpreviously by us (Nakatsura, T et al., Clin Cancer Res 10, 8630-8640,2004). Thus the obtained BM-DC were cultured with a mixture of the 4types of peptides selected as described above at a concentration of 10μM for 2 hours, and the peptide-pulsed BM-DC (5×10⁵ cells) were thenintraperitoneally administered to each mouse. After an interval of 1week, the same peptide-pulsed BM-DC were administered to the mousetwice, so that the mouse was strongly immunized. Thereafter, the spleencells of the mouse were recovered and induction of killer T cells wasevaluated. In order to strictly analyze induction of killer T cellsderived from CD8⁺ T cells, after collection of the spleen, CD4⁺ T cellswere eliminated from the collected spleen cells, using MACS beads, andthe remaining cells were then used for analysis.

The following protocols were used for generation and characterization ofSPARC-specific killer T cells (The day of recovering the spleen cellsfrom the immunized mouse was defined as Day 0.)

Day-21 (1) GM-CSF was added to the bone marrow cells of a BALB/c mouse,so as to start induction of bone marrow-derived dendritic cells(hereinafter referred to as BM-DC).

Day-14 (2) A mixture of the 4 types of SPARC peptides was added to theinduced BM-DC, and 2 hours later, 5×10⁵ cells were intraperitoneallyadministered to each mouse.

Steps (1) and (2) were repeated twice every other week.

Day 0 The spleen cells of the immunized BALB/c mouse were recovered. Thecells were co-cultured with BM-DC prepulsed with each SPARC peptide for2 hours. Thereafter, the resultant cells was co-cultured for 6 days.

Day 6 In order to detect killer T cells specifically recognizing theSPARC peptides, a Cr-release assay was carried out. T2K^(d) cells, an RLmale 1 cell line, a Meth A cell line, and a BALB/3T3 cell line wereselected as target tumor cells for killer T cells.

(4) Analysis of Cytotoxic Effects of SPARC-Specific Killer T Cells onTumor Cells by Cr-Release Assay

The cytotoxic activity of the induced SPARC-specific killer T cells ontumor cells was analyzed as follows. T2K^(d) cell is a cell lineobtained by introducing a K^(d) gene into a mouse T2 cell line thatlacks the expression of a TAP gene. Only in a case when a peptide addedfrom outside binds to an MHC class I molecule due to TAP deficiency, theexpression of a complex of the MHC class I molecule and the peptide isstably expressed on the cell surface. An RL male 1 cell line is BALB-cmouse-derived T-cell leukemic cells and does not express SPARC. Theabove two types of cells do not express SPARC. On the other hand, a MethA cell line and a BALB/3T3 cell line are BALB/c mouse-derivedfibrosarcoma cell line and fibroblast cell line, respectively. Both ofthese cell lines express SPARC. The aforementioned cell lines werelabeled with radioactive chromium (Cr), and they were then cultured withthe aforementioned killer T cells for 4 hours. Thereafter, a culturesupernatant was recovered, and the amount of radioactive Cr releasedfrom dead cells was quantified, so that cytotoxic activity was detected.

As a result, no cytotoxicity to T2K^(d) cells and RL male 1 cells, whichdid not express SPARC, was observed. However, cytotoxicity was observedspecifically to the T2K^(d) cells, which expressed a SPARC peptides inthe context of the K^(d) molecule on the surface by loading the SPARCK^(d) peptides 1, 3 and 4, and also to Meth A and BALB/3T3, whichspontaneously expressed SPARC (FIG. 3).

Example 3 (5) Induction of Antitumor Immunity in BALB/c Mice byImmunization with SPARC Peptide

(Method)

BM-DC was cultured with a mixture of SPARC K^(d)-1, 3 and 4 peptides (10μM for each) for 2 hours. Thereafter, 5×10⁵ cells were intraperitoneallyadministered to each mouse. After an interval of 1 week, apeptide-loaded BM-DC was administered to the mouse in the same manner,so that the mouse was immunized two times in total. Seven days later, amouse fibrosarcoma cell line, Meth A (1×10⁶ cells), which highly expressmouse SPARC, was subcutaneously transplanted to the mouse. Thus, thegrowth of the tumor and the survival time of the mouse were examined.

(Results)

The results are shown in FIG. 4. Preventive administration of SPARCpeptide pulsed BM-DC could induced inhibition of the growth of thesubcutaneously transplanted Meth A and induced a prolongation of thesurvival time of the mouse.

INDUSTRIAL APPLICABILITY

HLA-A24 (A*2402) is the most frequent HLA class I allele, possessed byapproximately 60% of the Japanese. The structural motifs of peptidesbound to the K^(d) molecules of BALB/c mouse is significantly similar tothose of peptides bound to the human HLA-A24 molecules. Accordingly, itwas revealed that, when a BALB/c mouse is immunized with a certainpeptide, if the peptide binds to the K^(d) molecule and thereby induceskiller T cells, and if the killer T cells destroy cancer cells thatexpress a complex of the peptide and the K^(d) molecule, it is highlypossible that the peptide also binds to HLA-A24 and induces human killerT cells that can destroy cancer cells. Therefore, the peptide of thepresent invention can be applicable to an immunotherapy for patientswith diffuse infiltrative stomach cancer, pancreatic cancer andmelanoma, who possess HLA-A24. It is anticipated that the QOL of thepatients can be improved by suppressing the growth or progression ofsuch cancers by SPARC peptide-based cancer immunotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the degree of expression of the top 15 genes that wereexpressed in stomach cancer tissues at a level higher than in normalstomach mucosal membrane, which were identified by performing cDNAmicroarray analysis on 20 patients with diffuse infiltrative stomachcancer. Taking into consideration the obtained results as well as theresults of the cDNA microarray analysis of normal tissues as describedbelow, from the aforementioned 15 genes, secreted protein acidic andrich in cysteine (SPARC) specifically overexpressed in a canceroustissue was selected as the most ideal cancer-specific antigen in diffuseinfiltrative stomach cancer.

FIG. 2 shows the results obtained by analyzing the expression of SPARCin 25 types of main adult organs and in 4 types of embryonal organs,using a cDNA microarray. The expression of the SPARC gene was observedeven in some normal tissues, but the expression level in such normaltissues was significantly lower than that in a cancerous tissue.

FIG. 3 shows the cytotoxicity of SPARC-positive tumor cells by CTL whichwas induced using a mouse K^(d)-restricted SPARC K^(d)-1, K^(d)-3 orK^(d)-4 epitope peptide in BALB/c mice.

FIG. 4 shows the suppression of the growth of the subcutaneouslytransplanted Meth A cancer cells and the extension of the survival timeof BALB/c mice by pre-administration of bone marrow-derived dendriticcells (BM-DC) loaded with a mixture of SPARC K^(d)-1, K^(d)-3 andK^(d)-4 peptides.

1. An isolated peptide of any of the following: (A) a peptide whichconsists of the amino acid sequence as shown in SEQ ID NO: 3; or (B) apeptide which consists of the amino acid sequence as shown in SEQ ID NO:3 in which one to three amino acids have been substituted or added, andwhich has capacity to induce cytotoxic (killer) T cells.
 2. Acomposition which comprises at least one type of the peptide of claim 1.3. An isolated antigen-presenting cell, which comprises a complex of anHLA molecule and the peptide of claim 1.